Cloning provides the foundation for medical advances, including genetic fingerprinting, amplification, and alteration of DNA. However, cloning also raises ethical concerns.
In molecular cloning, restriction enzymes are used to cleave DNA at specific locations, leaving complementary ‘sticky’ ends that can be joined by ligase. NEB developed a set of one-buffer systems compatible with many common restriction enzymes, allowing investigators to perform “diagnostic digests.”
Recombinant Proteins
Molecular cloning has made it possible to create and produce proteins that can be useful in a wide variety of applications. Many of these proteins have biopharmaceutical uses, including insulin, which treats diabetes; human growth hormone, which boosts growth in children and teenagers; and tissue plasminogen activator, which prevents blood clots after strokes or heart attacks. Other proteins produced through molecular cloning are used in diagnostic testing, food production, and research tools to study gene structure and function.
Early DNA cloning methods were laborious and time-consuming. Restriction enzyme preparations were often unreliable due to inefficient purification procedures, and plasmid vectors that could be used for cloning were cumbersome and difficult to work with. Scientists eventually developed the first standardized bacterial vector for cloning, the pBR322 plasmid. This vector was small, 4 kilobases in size, and contained essential features for cloning, such as an origin of replication, a selectable marker gene (e.g., antibiotic resistance), and a multiple cloning site for insertion of foreign DNA sequences. In addition, this plasmid-encoded an essential portion of the beta-galactosidase coding sequence, which allowed scientists to distinguish colonies that contained recombinant DNA from those that did not.
Today, most molecular cloning experiments begin with the insertion of a foreign DNA sequence into a specialized plasmid or viral vector. This vector is designed to contain additional sequences, such as a strong promoter, translation initiation signals, regulatory elements, and resistance markers, which allow the recombinant DNA to be expressed in the host organism of choice.
Once the recombinant DNA has been inserted into the vector, it is transformed into the host organism and replicated through natural DNA-replication processes and cell division. In most cases, specialized DNA-expression vectors are chosen that can be easily transformed into the host organism of interest for optimal results.
The creation of recombinant DNA sequences with desired characteristics begins with a PCR reaction. Template DNA, which contains the desired genetic sequence, is mixed with primers (short pieces of complementary single-stranded DNA), a polymerase enzyme that copies DNA, and a DNA ligase that covalently joins the ends of the DNA chain together. The resulting DNA mixture is then treated with uracil DNA glycosidase, which substitutes the deoxythymidine bases with uridines, and endonuclease VIII, which removes the uracils, leaving a 3′ overlap that can be annealed to a corresponding DNA fragment of the target sequence.
Vaccines
Making multiple copies of a particular DNA sequence allows scientists to study genes in more detail and engineer organisms with desired characteristics. This process is used in medicine, agriculture, and a variety of other applications. In medicine, cloning techniques are used to create vaccines that prevent or treat diseases. Cloning is also important for the synthesis of vitamins, hormones, and antibiotics. In agriculture, cloned bacteria enable nitrogen fixation in plants and other benefits.
The basic DNA cloning technique involves inserting the target gene into a circular piece of DNA called a plasmid. Plasmids replicate independently of bacterial chromosomal DNA and contain unique restriction sites that allow the insertion of foreign DNA fragments. The plasmid is then introduced into bacteria, and the inserted DNA fragment is copied as bacteria grow. The resulting recombinant plasmid contains the targeted gene and is used for further study or to produce a protein product.
To clone a DNA fragment, scientists first use restriction enzymes to cut it at specific sequences. They then use DNA ligase to join the ends of the DNA fragment together. The resulting recombinant DNA is then introduced into a plasmid-containing strain of bacteria. As the bacteria reproduce, they copy the plasmid and its recombinant gene, and the recombinant gene is expressed as proteins.
A specialized vector may be used instead of a plasmid for some specialized applications. For example, if scientists want to express the recombinant gene in a different host organism, they will use a “shuttle vector” that contains signals for transcription and translation in that host species.
Another major application of DNA cloning is in gene therapy, which involves replacing a disease-causing gene with a normal one. For example, scientists have successfully inserted a normal copy of the gene that causes cystic fibrosis into embryonic stem cells. These stem cells can then develop into many different types of cells and tissues, including nerve cells to repair damaged spinal cords and insulin-making cells to treat diabetes.
Gene Therapy
The ability to clone genes provides scientists with an enormous resource for basic and applied biological research. For example, cloned DNA sequences can be used as templates for making artificial proteins. These proteins can then be subjected to a variety of biochemical tests to determine their structure and function. Cloned DNA sequences can also be used to make antibodies against specific antigens. This is useful for developing vaccines to fight diseases such as hepatitis B, cholera, and influenza A.
Another major application of DNA cloning is the production of genetically engineered human cells to treat medical conditions such as cancer, diabetes, and hemophilia. In this therapy, a section of DNA containing the instructions for making a useful protein is packaged in a vector, such as a virus, bacteria, or plasmid. The vector is then used to deliver the gene into the cells of a patient with a diseased condition. This method has been successfully used to treat some genetic diseases, including adenosine deaminase severe combined immunodeficiency, Leber’s congenital amaurosis, and lysosomal storage diseases such as X-linked chronic granulomatous disease.
Molecular cloning has also been used to develop synthetic, artificial DNA that can be used to model how natural genes work in the body. This can be done by inserting a desired DNA fragment into a plasmid and then using restriction enzymes to cut and seal the ends of the DNA together. Then, DNA ligase can covalently link the DNA fragments together to form the artificial DNA.
Cloning technology has also been applied to creating recombinant antigens for developing vaccines against hepatitis B, cholera, influenza A, and other infectious agents. These recombinant antigens have replaced the use of live viruses in developing modern vaccines and are now used worldwide for the manufacture of hepatitis B, cholera, flu, polio, and other vaccines.
Molecular cloning has led to the development of techniques for assembling multiple DNA fragments into a single, continuous stretch of DNA called a gene construct. These new methods allow the cloning of genes into a vector, the creation of recombinant proteins, and the production of recombinant antigens for the manufacture of vaccines and other therapeutic agents.
Cell Therapy
Molecular cloning techniques have allowed scientists to produce large numbers of identical copies of a particular DNA segment. This ability to make many copies of a sequence makes it much easier to study the gene’s role in a given biological process. It also makes it possible to create recombinant proteins for research purposes. Molecular cloning techniques have also enabled the development of vaccines and other treatments that are based on DNA segments.
The first step in cloning is to isolate the DNA fragment to be copied. This can be done by digesting the DNA with restriction enzymes or by using PCR to amplify and target the DNA fragment. The DNA fragment is then combined with the vector DNA in a reaction called ligation, in which a ligase enzyme joins together the complementary strands.
A ligation reaction is more efficient when the DNA fragments used are of a similar size. For this reason, a great deal of effort has been put into developing new technologies to allow DNA fragments to be assembled seamlessly. These techniques can turn a series of independent DNA digestion, restriction, and ligation reactions into a single tube-based procedure.
Genomic DNA can be cloned from prokaryotes (cells without a nucleus) or eukaryotes, such as human cells. Eukaryotic genomes have introns, which are regions of the DNA that do not code for a protein, and eukaryotic genes have regulatory signals that differ from those of prokaryotes. In order to clone a gene from a eukaryotic organism, a DNA library is first grown in a collection of Petri dishes. A porous membrane is then placed over the top of each plate, and a solution of DNA labeled with a radioactive probe is bathed on the membrane. The radioactive probe sticks to any DNA fragments that have a matching sequence. The membrane is then rinsed, blotted, and affixed to a sheet of radiation-sensitive film.
Once a cloned stretch of DNA has been isolated, it can be sequenced to determine its specific chemical composition. This information is then used to design oligonucleotides, which are short pieces of complementary DNA that can be used in further experiments. Cloning has also enabled scientists to identify new genes and study the effect of mutations on a particular gene. This type of experiment is what led to the creation of Dolly, the first mammal to be cloned by nuclear transfer.
